Biomedical subjects

Doo-Hong Park

Publications and source records attributed to Doo-Hong Park.

5 recordsLinked to original sources

PubMed2004

Participation of histidine-51 in catalysis by horse liver alcohol dehydrogenase.

Histidine-51 in horse liver alcohol dehydrogenase (ADH) is part of a hydrogen-bonded system that appears to facilitate deprotonation of the hydroxyl group of water or alcohol ligated to the catalytic zinc. The contribution of His-51 to catalysis was studied by characterizing ADH with His-51 substituted with Gln (H51Q). The steady-state kinetic constants for ethanol oxidation and acetaldehyde reduction at pH 8 are similar for wild-type and H51Q enzymes. In contrast, the H51Q substitution significantly shifts the pH dependencies for steady-state and transient reactions and decreases by 11-fold the rate constant for the transient oxidation of ethanol at pH 8. Modest substrate deuterium isotope effects indicate that hydride transfer only partially limits the transient oxidation and turnover. Transient data show that the H51Q substitution significantly decreases the rate of isomerization of the enzyme-NAD(+) complex and becomes a limiting step for ethanol oxidation. Isomerization of the enzyme-NAD(+) complex is rate limiting for acetaldehyde reduction catalyzed by the wild-type enzyme, but release of alcohol is limiting for the H51Q enzyme. X-ray crystallography of doubly substituted His51Gln:Lys228Arg ADH complexed with NAD(+) and 2,3- or 2,4-difluorobenzyl alcohol shows that Gln-51 isosterically replaces histidine in interactions with the nicotinamide ribose of the coenzyme and that Arg-228 interacts with the adenosine monophosphate of the coenzyme without affecting the protein conformation. The difluorobenzyl alcohols bind in one conformation. His-51 participates in, but is not essential for, proton transfers in the mechanism.

Alcohol Dehydrogenase↗

PubMed2004

Improved recombinant gene expression in CHO cells using matrix attachment regions.

The use of animal cells such as Chinese hamster ovary (CHO) cells for recombinant gene expression provides many advantageous features such as proper folding and post-translational modification of the recombinant protein. However, recombinant genes introduced into animal cells are often expressed at low levels mainly due to position effects from the neighboring chromatin context. The tedious and time-consuming selection and amplification procedure has been the major hurdle for using animal cell line such as CHO cells. To improve mammalian cell expression systems, we screened a variety of matrix/scaffold attachment region (MAR/SAR) elements for their ability to insulate transgene expression from the position effects in CHO cells. We found that the human beta-globin MAR element is particularly effective as the frequency of beta-Gal positive colonies was increased by up to 80%. The expression levels of these colonies were also enhanced about seven-fold. These improvements appear to be related to the increased copy numbers and a higher efficiency of expression of the integrated genes. When this element was used to express soluble TGF-beta type II receptor (sTbetaRII) through the gene amplification system, the frequency of colonies expressing detectable amounts of sTbetaRII was much higher than that of the control vector. We could also generate high sTbetaRII producers with uniform growth properties by a simple two-step amplification process involving two concentrations of methotrexate. This eliminates the need to isolate individual colonies followed by multi-step treatments of methotrexate and thereby greatly simplifies this mammalian expression system.

Animals↗

PubMed2004

Purification and molecular cloning of a novel serine protease from the centipede, Scolopendra subspinipes mutilans.

A novel serine protease, named as scolonase, was purified and characterized from the tissue of the Korean centipede, Scolopendra subspinipes mutilans. Purified scolonase showed an apparent molecular weight of 25 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and an isoelectric point of 4.8 on isoelectric focusing gel. Scolonase was able to preferentially hydrolyze arginine over lysine at the cleavage site among the several synthetic peptide substrates. Scolonase has also a potent fibrinolytic activity by converting human Glu-plasminogen to activated plasmin due to the specific cleavage of the molecule at the peptide bond Arg(561)-Val(562). The enzyme activity of scolonase was completely inhibited by phenylmethanesulfonyl fluoride and difluorophosphate. The cDNA encoding scolonase was cloned from the cDNA library of the centipede constructed with oligonucleotide probe, which was designed on the basis of the N-terminal amino acid sequence of scolonase. The deduced complete amino acid sequence of scolonase demonstrated that the protein is composed of 277 amino acids including 33 amino acids as a leader sequence, and that it has significant sequence homology with other serine proteases.

Amino Acid Sequence↗

PubMed2004

Characterization and biological activities of recombinant human plasminogen kringle 1-3 produced in Escherichia coli.

Angiogenesis, the formation of new capillaries from preexisting blood vessels, is involved in many pathological conditions, for example, tumorigenesis, diabetic retinopathy, and rheumatoid arthritis. Angiostatin, which contains the kringle 1-4 domains of plasminogen, is known to be a potent inhibitor of angiogenesis and a strong suppressor of various solid tumors. In this study, we expressed recombinant protein containing the kringle 1-3 domains of human plasminogen in Escherichia coli and investigated its biological activities. The protein was successfully refolded from inclusion bodies and purified at a 30% overall yield, as a single peak by HPLC. The purified recombinant protein had biochemical properties that were similar to those of the native form, which included molecular size, lysine-binding capacity, and immunoreactivity with a specific antibody. The recombinant protein was also found to strongly inhibit the proliferation of bovine capillary endothelial cells in vitro, and the formation of new capillaries on chick embryos. In addition, it suppressed the growth of primary Lewis lung carcinoma and B16 melanoma in an in vivo mouse model. Our findings suggest that the recombinant kringle 1-3 domains in a prokaryote expression system have anti-angiogenic activities, which may be useful in clinical and basic research in the field of angiogenesis.

Amino Acid Sequence↗

PubMed2003

Inhibition of angiogenesis and angiogenesis-dependent tumor growth by the cryptic kringle fragments of human apolipoprotein(a).

Apolipoprotein(a) (apo(a)) contains tandemly repeated kringle domains that are closely related to plasminogen kringle 4, followed by a single kringle 5-like domain and an inactive protease-like domain. Recently, the anti-angiogenic activities of apo(a) have been demonstrated both in vitro and in vivo. However, its effects on tumor angiogenesis and the underlying mechanisms involved have not been fully elucidated. To evaluate the anti-angiogenic and anti-tumor activities of the apo(a) kringle domains and to elucidate their mechanism of action, we expressed the last three kringle domains of apo(a), KIV-9, KIV-10, and KV, in Escherichia coli. The resultant recombinant protein, termed rhLK68, exhibited a dose-dependent inhibition of basic fibroblast growth factor-stimulated human umbilical vein endothelial cell proliferation and migration in vitro and inhibited the neovascularization in chick chorioallantoic membranes in vivo. The ability of rhLK68 to abrogate the activation of extracellular signal-regulated kinases appears to be responsible for rhLK68-mediated anti-angiogenesis. Furthermore, systemic administration of rhLK68 suppressed human lung (A549) and colon (HCT-15) tumor growth in nude mice. Immunohistochemical examination and in situ hybridization analysis of the tumors showed a significant decrease in the number of blood vessels and the reduced expression of vascular endothelial growth factor, basic fibroblast growth factor, and angiogenin, indicating that suppression of angiogenesis may have played a significant role in the inhibition of tumor growth. Collectively, these results suggest that a truncated apo(a), rhLK68, is a potent anti-angiogenic and anti-tumor molecule.

Allantois↗